Diabetes mullitus is the most prevalent metabolic disease of humans. The most common form of diabetes is associated with peripheral insulin resistance, a condition whereby muscle tissue fails to respond to elevated circulating insulin with the appropriate uptake and disposal of blood glucose. Recent evidence indicates that insulin resistance results from a disruption in the normal mechanism by which insulin activates glucose transport in skeletal muscle. In a previous grant period, we identified and cloned Glut4, one of at least 10 known glucose transporter (Glut) isoforms. Glut4 is a key rate-limiting component in insulin-stimulated skeletal muscle glucose uptake. The long-term goal of this proposal is to elucidate the molecular mechanisms involved in the regulation of Glut4 and other Glut isoforms and to identify defects in these processes that predispose to diabetes mellitus or other metabolic disorders. The following specific aims will be undertaken in the next project period: 1. Insulin stimulates glucose transport in fat and muscle by altering the subcellular traffcking of Glut4 and causing its redistribution frorn intracellular membrane compartments to the cell surface. At least 3 distinct targeting motifs have been identified in Glut4 that regulate its subcellular trafficking in adipocytes, although nothing is known about their targeting functions in skeletal muscle. The precise role of these motifs in the subcelluIar traffcking of Glut4 in muscles cells will be explored via site-directed mutagenesis in conjunction with an adenoviral system that allows the analysis of recombinant proteins expressed in L6 myoblasts and intact rodent skeletal muscle. 2. Syncrip has been identified in adipocytes as a RNA-binding protein associated with low-density microsomes that becomes phosphorylated on tyrosine residues in response to insulin. Syncrip is also known to bind to Synaptotagmins, proteins involved in the regulation of vesicle fusion at the plasma membrane in neuroendocrine cells. The possible role of Syncrip in regulating insulin-stimulated protein secretion and Glut4 translocation in adipocytes will be investigated. 3. A novel mammalian Glut isoform whose expression may be restricted to muscle and heart has recently been discovered via the analysis of human EST and genomic sequence data. This isoform will be thoroughly characterized with respect to its substrate specificity, tissue distribution, regulation, and possible role in insulin regulated metabolite uptake.